Barley endoproteolytic enzymes are important to germination because they hydrolyze endosperm storage proteins to provide precursors for new protein synthesis. We recently developed an electrophoretic method utilizing gel-incorporated protein substrates to study the endoproteinases of 4-d-germinated barley (Hordeum vulgare L. cv Morex) grain. This work extends those findings to determine the temporal pattern of the appearance of the endoproteinases during germination, the sensitivities of the proteinases to class-specific proteinase inhibitors, and where, in germinating caryopses, the proteinases reside. Six endoproteinase activity bands (representing a minimum of seven enzymes) were present in 5-d-germinated barley grain extracts subjected to electrophoresis in nondenaturing gels at pH 8.8. The activities of two of the enzyme bands ("neutral" proteinases) increased as the pH was increased from 3.8 to 6.5. The activities of the remaining four ("acidic") bands diminished abruptly as the pH increased above 4.7. Two proteinase bands hydrolyzed gelatin but not edestin, four of the proteinases hydrolyzed both gelatin and edestin at nearly the same rates, and one enzyme degraded only edestin. One neutral endoproteinase was sensitive to diisopropyl fluorophosphate inhibition, and the other was not inhibited by any of inhibitors tested. Four of acidic enzymes were cysteine proteinases linhibited by trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and Nethylmaleimide]; the other was an aspartic acid endoproteinase (sensitive to pepstatin). Only the aspartic proteinase was detected in either ungerminated or steeped barley grain. During the germination (malting) process, the aspartic endoproteinase activity decreased until the second day of germination and then increased until germination day 5. The first endoproteinase(s) induced during germination was a neutral enzyme that showed activity on the 1st day of the germination phase after steeping. Most of the endoproteinases became active on the 2nd or 3rd germination day, but one cysteine proteinase was not detected until the 5th day. Acid cysteine proteinases were present in the aleurone, scutellum, and endosperm tissues but not in shoots and roots. The aleurone layer and endosperm contained almost exclusively band B1 neutral proteinases, whereas the scutellum, shoots, and roots contained both Bl and B2 bands. This work shows that germinating barley contains a complex set of proteinases whose expression is temporally and spatially controlled. But, at the same time, it also shows that this electrophoretic method for separating and studying individual enzymes of this complex will allow us to more readily characterize and purify them.

No abstract

The endoproteolytic enzymes of malt influence several different aspects of malt and beer quality. For this reason, we are extracting and characterizing these enzymes from green malt. The proteolytic activity of a Morex green malt extract was highest at pH 3.8 with haemoglobin substrate but gelatin hydrolysis was maximal from pH 4.7 to 6.0. Endoproteolytic hydrolysis of a 55% isopropanol-soluble reduced hordein fraction was about three times slower than gelatin hydrolysis but was relatively constant over the pH range from 3.8 to 6.5, although the activity did decrease at more acidic (3.0) or basic (7.0) pH values. To study the green malt proteinases in detail, a non-denaturing electrophoretic system was developed in which substrate proteins-either gelatin, edestin or hordein-were incorpor ated into an electrophoretic gel. After electrophoresis and incubation of the gels at pH 3.8,4.7, 5.5, or 6.5 to allow enzymatic hydrolysis, the separated activities were determined by using protein staining to determine where the incorporated substrate had been hydrolysed. Using this system, seven proteolytic activity bands were detected. Five of the bands were maximally active at pH 3.8 and their activities dropped quickly as the pH increased. The other two bands, which migrated more slowly, hydrolysed gelatin more rapidly than they did the other substrates tested. Their gelatinolytic activities increased as the pH was raised (by 3-to 6-fold in the pH range tested).The electrophoretic system described has proven very useful for studying the proteinases of germinat ing barley seeds. The results indicate that much past research on malt proteinases may not be particu larly relevant to the malting and brewing industries because it was conducted under pH conditions and with substrates that are likely quite different from those in the seed during the barley germination process. By using electrophoresis to separate proteinases before analysis, we can now study their individual characteristics and thus can conduct studies more relevant to malting and brewing.

A competitive inhibition radioassay of thiamine is described using a gel obtained by coupling a buckwheat-seed thiamine-binding protein to CNBr-activated Sepharose. The sample to be analysed is incubated with gel suspension and [14C]thiamine and after centrifugation the radioactivity of the supernatant is measured. The method is simple and specific, and applicable over a thiamine concentration range 0.5-5 microM with a coefficient of variation typically below 5%. The gel is reusable and stable for several months. Applicability of the method for direct determination of thiamine in multivitamin pharmaceuticals is demonstrated.